The RNA m5C modification in R-loops as an off switch of Alt-NHEJ.

The roles of R-loops and RNA modifications in homologous recombination (HR) and other DNA double-stranded break (DSB) repair pathways remain poorly understood. Here, we find that DNA damage-induced RNA methyl-5-cytosine (m5C) modification in R-loops plays a crucial role to regulate PARP1-mediated poly ADP-ribosylation (PARylation) and the choice of DSB repair pathways at sites of R-loops. Through bisulfite sequencing, we discover that the methyltransferase TRDMT1 preferentially generates m5C after DNA damage in R-loops across the genome. In the absence of m5C, R-loops activate PARP1-mediated PARylation both in vitro and in cells. Concurrently, m5C promotes transcription-coupled HR (TC-HR) while suppressing PARP1-dependent alternative non-homologous end joining (Alt-NHEJ), favoring TC-HR over Alt-NHEJ in transcribed regions as the preferred repair pathway. Importantly, simultaneous disruption of both TC-HR and Alt-NHEJ with TRDMT1 and PARP or Polymerase θ inhibitors prevents alternative DSB repair and exhibits synergistic cytotoxic effects on cancer cells, suggesting an effective strategy to exploit genomic instability in cancer therapy.

DNA hypomethylation silences anti-tumor immune genes in early prostate cancer and CTCs.

Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.